PROJECT III: FUNCTIONAL EVALUATION OF CDH PATIENT GENE VARIANTS IN VITRO AND IN SELECT ANIMAL MODELS ABSTRACT Significant progress has been made in the care of Congenital Diaphragmatic Hernia (CDH) patients in the past two decades. Survival rates have been steadily improving, in part because of gentle ventilation techniques, high-frequency ventilation, cardiovascular pharmacologic support, and ECMO. Currently, the main therapeutic goal is to stabilize the cardiac and respiratory systems in CDH infants, while attempting to minimize iatrogenic (therapy-induced) injury to the delicate pulmonary tissue, before undergoing surgical repair of the diaphragm. Lengthy stays in the neonatal intensive-care unit (NICU) are often necessary. Despite these successes, a large number of infants still succumb to the pulmonary complications of CDH, with late deaths mostly due to pulmonary hypertension and pulmonary insufficiency. A major pulmonary defect associated with CDH is insufficient gas exchange, and pathology sections indicate that this is likely due to reduced number of alveolar septa. At the same time, a major contribution to lethality of infants with CDH is severe pulmonary hypertension due to abnormal vascular and vascular smooth muscle development, as well as failure of the vascular smooth muscle to relax normally after birth. Using a complementary set of mammalian genetic, genomic, physiological, and three-dimensional morphological approaches, we propose to characterize in vitro and in vivo the pulmonary defects of CDH model organisms focusing on postnatal timing and cell types, including myofibroblasts and extracellular matrix constituents such as elastin. By identifying molecular mechanisms affected by different genetic mutations, we will design molecular assays that could eventually provide new paradigms for therapy, and reduce the economic, societal, and personal burden of CDH.